The invention relates to a method for the controlled, limited proteolytic cleavage of proteins, in particular proenzymes, for recovering enzymes and protein fragments, respectively, wherein the protein is treated with a protease. In particular, the invention relates to a method of recovering thrombin from prothrombin.
Such a method has been known from EP-A-0 378 798. According to that method, prothrombin derived from plasma or a plasma fraction is adsorbed on a solid carrier (immobilized), and the adsorbate is treated with Ca.sup.2+ ions. The Ca.sup.2+ ions are used in concentrations up to 30 mM and, together with the proteases also derived from the plasma and adsorbed on the carrier, cause the thrombin to be cleaved from the adsorbate. As the solid carrier, methacrylic and acrylic copolymers are used.
It has furthermore been known that trypsin degrades dissolved prothrombin into fragments of low molecular weight, wherein a complete loss of the thrombin activity is to be found (Biochim. Biophys. Act. 329, 221-232 (1973)); the proteolytic degradation of the prothrombin thus does not end with thrombin, and thus this method does not lend itself to the recovery of thrombin.
All the methods of the initially defined kind lead to different prothrombin cleavage products, depending on the protease and treatment conditions used, which cleavage products in part may be used therapeutically (e.g. thrombin) and in part may be used diagnostically and for the recovery of specific antibodies. For all these applications it is, however, necessary that the protein fragments are highly pure, which necessarily involves a lot of work, since all the known cleavage methods lead to a plurality of fragments. A further disadvantage of these multi-stage and time-consuming purification methods is that they necessarily involve high losses of yield.